1. Technical Field
The present invention relates to is pharmaceuticals and especially radiopharmaceuticals for use as diagnostic and therapeutic agents. More specifically, the present invention relates to compounds and methods of synthesizing compounds which utilize multi-dentate ligands which form stable complexes with metal compounds both with and without the need of external reducing agents for use as diagnostic or therapeutic radiopharmaceuticals.
2. Background Art
Because of the favorable physical properties, widespread availability, and low cost of .sup.99m Tc, this radionuclide continues to be the most attractive candidate to formulate diagnostic radiopharmaceuticals for scintigraphic imaging studies in patients (Jurisson et al., 1993). Re, a chemical analogue of Tc, has two radioisotopes (i.e., .sup.186 Re and .sup.188 Re; .sup.186/188 Re) that have physical and production properties that make them among the most attractive beta-emitting radionuclides for formulation of new therapeutic radiopharmaceuticals (Volkert et al., 1991; Troutner, 1987). Since the chemical properties of Tc and Re are often identical (although, not always) many ligand systems can be used as a basis to synthesize bifunctional chelating agents (BFCAs) that are capable of forming chelates with .sup.99m Tc that have the same structural and physicochemical properties as the corresponding .sup.186/188 Re chelates.
Development of sophisticated molecular probes in the design of new .sup.99m Tc- and .sup.186/188 Re radiopharmaceuticals will provide for future advances in the diagnosis and treatment of patients. While many important single photon emission computed tomography (SPECT) radiopharmaceuticals are effectively used as specific tools for diagnosis of human disease, accelerated development of many new site-directed synthetic derivatives (e.g., immunologically derived molecules, receptor-avid molecules, etc.) will provide a multitude of opportunities for further technological advances for both diagnostic and therapeutic applications.
When developing effective site specific therapeutic or diagnostic radiopharmaceuticals, many important factors must be considered. It is essential that the metallic radionuclide (e.g. Re-188 or Tc-99m), upon interaction with a bifunctional chelating agent, should form an in vivo stable complex in high specific activities with 1:1 metal to ligand stoichiometry. These stringent requirements restrict the choice to only a few ligand backbones and, therefore, necessitates the design and development of new bifunctional chelating agents. Most importantly, a detailed understanding of the coordination chemistry of new ligand systems with non radioactive rhenium is important for the subsequent extention of these reactions at the tracer levels to label bifunctional chelating agents using Re-188.
Many difficulties encountered in the design of highly selective radiolabeled drug carriers must be overcome (e.g., problems in efficient drug delivery to target sites, in vivo metabolism, rates of clearance of radioactivity from non-target tissues relative to target tissues, etc.). The physicochemical characteristics of the .sup.99m Tc- and .sup.186/188 Re-chelate moiety attached or fused to the site-directed molecule will play a crucial role as an inherent determinant of the effectiveness of the final drug product. In addition, the ability of .sup.99m Tc or .sup.186/188 Re to label the final product under conditions amenable for routine formulation of radiopharmaceuticals is also an essential consideration.
Labeling of biomolecules with .sup.99m Tc or .sup.186/188 Re to produce effective radiopharmaceuticals presents many challenges. It is necessary to produce .sup.99m Tc and/or .sup.186/188 Re labeled drugs that have high in vitro and in vivo stabilities. Several different ligand frameworks have been developed that form .sup.99m Tc or Re chelates exhibiting minimal or no measurable in vivo or in vitro dissociation. These chelates have provided radiopharmaceutical chemists with a selection of .sup.99m Tc-chelates that have a range of physicochemical characteristics.
The formation of .sup.99m Tc (viz Re) products in high yields with high radiochemical purity (RCP), however, usually requires the presence of large quantities of excess ligand during the formulation processes that are used for routine pharmaceutical preparation. Unfortunately, the high specific activities (i.e., GBq/.mu.mole or Ci/.mu.mole) required for radiolabeled site-directed synthetic derivatives being developed preclude the use of many of these chelation systems, thus, severely limiting the choice to only a few ligand backbones.
High specific activity (Sp. Act) radiolabeled agents can be prepared using either preformed .sup.99m Tc- or .sup.186/188 Re bifunctional chelates (BFCs) or post-conjugation chelation with the radioactive metals where a chelating moiety is already appended (Parker, 1990) or fused (Lister-James et al., 1994; Knight et al., 1994) to the biomolecular targeting agent. Even though maximization of Sp. Act can be achieved by separation of the radiolabeled from the non-radiolabeled molecules, practically, it is more desirable to employ chelation systems that require small quantities of the chelates. In the formation of products that will be ultimately used as FDA approved .sup.99m Tc/.sup.186/188 Re radiopharmaceuticals for routine patient care applications, it is most desirable to keep the number of steps for the formation of the drug-product to a minimum, ideally to one step, as is the case for most .sup.99m Tc- "instant kits".
One of the few ligand systems shown to be effective for preparation of high yield, stable .sup.99m Tc chelates using small quantities of chelator are the amido-thiol class of ligands (Fritzberg et al. 1988, Rao et al., 1992, and Chianelli et al, 1994). Generally, these types of multi-dentate ligands contain at least four donor atoms and one or two thiol donor groups in combination with two to three amido donor groups. Several N.sub.2 S.sub.2 or N.sub.3 S amido-thiol frameworks have been used to synthesize BFCAs and include diamidodithiol (DADS) ligands (Fritzberg et al., 1988), monoaminemonoamide (MAMA) ligands (Rao et al., 1992; Gustavson et al., 1991) and mercaptoacetylglycylglycylglycine (MAG.sub.3) ligands (Chianelli et al., 1994). While the amido-thiol ligands make effective BFCAs for .sup.99m Tc and .sup.186/188 Re, the range of their physicochemical properties are limited, conditions for routine labeling can be difficult to reduce to practical utility and external reducing agents (e.g., Sn(II) are usually present during labeling with .sup.99m Tc or .sup.186/188 Re which can cause irreversible alteration of the site-directed moiety reducing or eliminating specific in vivo localization.
Other ligand systems that have also been used for .sup.99m Tc labeling include N.sub.2 S.sub.2 -amine-thiol ligands, propylineamineoxime (PnAO) derivatives and the hydrazino nicotinamide (HYNIC) system. The former two derivatives form neutral lipophilic .sup.99m Tc-chelates, that while beneficial in some respects, result in high non-specific binding in vivo and poor clearance from non-target tissues (Muna et al., 1994; Noch et al., 1994). The HYNIC system does not form a well-defined product with .sup.99m Tc (Abrams et al., 1990a; Abrams et al., 1990b). All of these systems usually form chelates with .sup.99m Tc with the necessity of external reducing agents.
Ligand backbones containing trivalent phosphine donor groups have been shown to be effective in forming stable .sup.99m Tc and .sup.186/188 Re chelates in high RCP. Phosphines not only chelate .sup.99m Tc (or Re), but they are capable of reducing both pertechnetate and perrhenate to lower oxidation states, and, therefore, do not necessarily require the presence of an external reducing agent (e.g., Sn(II)).
Diphosphine ligands have been extensively used in the development of .sup.99m Tc-radiopharmaceuticals, particularly those that are used as .sup.99m Tc-labeled myocardial perfusion agents (Deutsch, 1993; Nowotnik and Nunn, 1992; Kelly et al., 1993). Unfortunately, most of these chelates utilize alkylphosphine donor groups and the phosphines are rapidly oxidized (to phosphorus oxides) in aqueous solutions containing O.sub.2 and require stringent conditions for manufacture of the drugs and for ultimate routine formation of the final product. For these reasons, ligands that contain alkyl phosphine donor groups have limited flexibility for the design of new drugs and do not form a rational basis to prepare most phosphine-based BFCAs for use in preparing site-directed radiopharmaceuticals.
Aromatic phosphines have also been reported for use with Tc and Re, however, the high lipophilicity of the resulting chelates minimize their potential utilization as BFCAs for in vivo applications.
A small ligand system containing phosphine donor groups with good solubility in aqueous solutions and not oxidized by O.sub.2, but still capable of reducing .sup.99m TcO.sub.4.sup.- or .sup.186/188 ReO.sub.4.sup.- and/or strongly chelating reduced Tc or Re, would find widespread applicability in formulating new radiopharmaceuticals or new BFCAs.
Bonding capabilities of phosphines with the early transition metals (e.g.; Technetium or Rhenium) are influenced not only by the .sigma. phosphorus-metal interaction which uses the lone pair of electrons on the P.sup.III center and a vacant orbital on the metal center, but also by the distinct possibility of synergic .pi. back-donation from a non-bonding d.pi. pair of electrons on the metal center into the vacant 3d.pi. orbital on the phosphorus. The .sigma. and .pi. bonds reinforce one another to produce strong phosphorus-metal bonds which are often stable even under in vivo conditions. (Greenwood and Earnshaw, 1993, Mayer and Kaska, 1994). Therefore, functionalized phosphines constitute an important family of ligands for use in nuclear medicine. For example, the Tc-99m based radiopharmaceuticals, Tetrafosmin and Technecard, which are currently being used as in vivo heart imaging agents, are derived from bis chelating and monochelating phosphines of the type (EtO(CH.sub.2).sub.2).sub.2 P(CH.sub.2).sub.2 P((CH.sub.2).sub.2 OEt) and P(CH.sub.2 CH.sub.2 OCH.sub.3).sub.3, respectively. (Higley et al., 1993, Jain et al., 1993, DeRosch et al., 1992, Marmion et al., 1995). While bis chelating phosphines of the DMPE class (where DMPE stands for 1,2-bis(dimethylphosphino)ethane) are able to produce in vivo stable Tc-99m complexes (Deutsch et al., 1981, Deutsch, 1993, Glavon et al., 1982, Vanderheyden et al., 1984, Vanderhyden et al., 1985), the inherent oxidative instability of DMPE and related alkyl phosphines limits their utility in terms of ligand backbone modifications to produce bifunctional chelating agents (BFCAs) in the development of Tc-99m (or Re186/188) labeled biomolecules. On the other hand, aryl phosphines are usually too large or highly charged (e.g. sulfonated aryl phosphines) and, therefore, may be unsuitable in the design of BFCAs for use in nuclear medicinal applications (Cornils and Wiebus, 1995). Studies by Deutsch et al., applicant, and several others have demonstrated that technetium (or rhenium) forms in vivo stable and kinetically inert bonds with phosphines. (DeRosch et al., 1992, Bandoli et al., 1984, Vanderheyden et al., 1985, Vanderheyden et al., 1984, Libson et al, 1983, Ichimura et al., 1984). Therefore, new developments in the design of phosphine ligands may aid in the discovery of new, performance effective, radiopharmaceuticals. In particular, the synthesis of functionalized phosphine frameworks that would result in the formation of Tc-99m or Re-188 complexes with 1:1 metal to ligand stoichiometrics becomes important in the context of design and development of radiopharmaceuticals produced via the labelling of specific biomolecules (e.g. peptides or proteins), for use in tumor specific diagnosis or therapy of human metastases. In this approach of designing diagnostic or therapeutic radiopharmaceuticals, it is important that the bifunctional chelating agent (ligand) be bound to a point of the biomolecule away from the active site (e.g. amino acid sequence necessary for receptor binding). Radiolabelling of the biomolecule/ligand complex with Tc-99m or Re-188 can then be carried out via strong covalent interactions of the metal center with specific donor atoms of the ligand, with no destruction of receptor specificity as is shown in FIG. 1. Simple aryl or alkyl functionalized phosphines (e.g. PPh.sub.3 or (H.sub.3 C).sub.2 PCH.sub.2 CH.sub.2 P(CH.sub.3).sub.2) produce strong and in vivo stable metal-phosphorus bonds. However, they are unsuited for use in the design of biomolecular labelled radiopharmaceuticals because, most often, the coordination chemistry of these ligands produces complexes with more than one ligand per metal center. The chemical modifications of (H.sub.3 C).sub.2 PCH.sub.2 CH.sub.2 P(CH.sub.3).sub.2 (DMPE) and other related alkyl phosphates present difficulties in forming complexes with one ligand per metal center. Furthermore, their oxidative instability and pyrophoric nature limit their use in the development of bifunctional chelating agents via ligand modification reactions. Several groups have investigated the coordination chemistry of technetium and rhenium with sulfur/nitrogen and phosphine containing ligands (Archer et al., 1995, Refosco et al., 1993, Tisato et al., 1995). However, the presence of bulky aryl substituents on the phosphines often limit their degree of solubility in aqueous solutions making them unsuitable for bifunctional chelating agents.
Most other bifunctional chelation systems require the presence of an external reducing agent (e.g., Sn.sup.+2) or prereduction of .sup.99m TcO.sub.4.sup.- or .sup.186/188 ReO.sub.4.sup.- to lower metal oxidation states (e.g. .sup.99m Tc-glucoheptonate). Water soluble phosphine groups containing low molecular side arms attached to each phosphine P-atom would provide versatility in ligand design and could be used as both as a reducing agent for .sup.99m TcO.sub.4.sup.- (or .sup.186/188 ReO.sub.4.sup.-), under conditions used for routine .sup.99m Tc-radiopharmaceutical preparation, and as an efficient complexing agent for the reduced forms of Tc or Re.
Applicants use a series of multi-dentate ligands containing functionalized hydroxyalkyl phosphines that are stable in aerated aqueous solutions and will form highly stable .sup.99m Tc and .sup.188 Re chelates. Unlike prior art alkyl phosphine based ligands designed to reduce or chelate .sup.99m Tc or .sup.186/188 Re, the hydroxyalkyl phosphine groups are not sensitive to the presence of oxygen when dissolved in aqueous solutions. Other water soluble phosphine ligands with good oxidative stability have also been used as reducing agents, however, the side chains attached to the phosphine donor P-atoms in these ligands are bulky and/or produce highly charged phosphines which limit their utility in radiopharmaceutical development (Pasqualine et al., 1994).
Most other bifunctional chelation systems require the presence of an external reducing agent (such as Sn(II) or NaBH.sub.4) or prereduction in order to reduce the .sup.99m TcO.sub.4.sup.- (or .sup.186/188 ReO.sub.4.sup.-) from the +7 oxidation state to lower oxidation states (e.g., .sup.99m Tc-GH) that are more readily chelated.
The ligands containing one or more hydroxyalkyl phosphine donor groups of the present invention require no external reducing agents, however, the ligand can be used as coordinating groups when used in conjunction with other reducing agents or .sup.99m Tc-synthons. The resulting .sup.99m Tc and Re complexes produced with these phosphine containing ligands exhibit excellent in vivo stability as well in aqueous solutions including human serum.